Part 1. We demonstrated that intra-mPFC administration of the selective KOR agonist U69,593 decreased local DA overflow, but failed to modify DAT function in 3H DA uptake studies. Reverse dialysis of the KOR antagonist nor-Binaltorphimine (nor-BNI) enhanced mPFC DA overflow. Extracellular amino acid levels were also affected by KORs, as evidenced by U69,593 inhibition of the ability of the glutamate reuptake blocker, l-trans-pyrrolidine-2,4-dicarboxylate to elevate mPFC glutamate and GABA levels. Whole-cell recordings from mPFC layer V pyramidal neurons revealed that U69,593 decreased the frequency, but not amplitude, of mini EPSPs, an effect occluded by picrotoxin. To determine whether KOR regulation of mPFC DA overflow was mediated by KOR on DA terminals, we utilized a Cre recombinase-driven mouse line lacking KOR in DA neurons. In these mice, basal DA release or uptake was unaltered relative to controls, but the attenuation of mPFC DA overflow by local U69,593 was not observed, indicating KOR acts directly on mPFC DA terminals to locally inhibit DA levels. Conditioning procedures were then used to determine whether mPFC KOR signaling was necessary for KOR-mediated aversion. U69,593-mediated conditioned place aversion was blocked by intra-mPFC nor-BNI microinjection. These findings demonstrate that mPFC KORs negatively regulate DA and amino acid neurotransmission, and are necessary for KOR-mediated aversion (manuscript submitted to the Journal of Neuroscience). Part 2. Previously we demonstrated that exposure to a cocaine-associated environment increases mPFC GABA transmission by enhancing excitatory drive from the BLA and activation of AMPA/KA receptors on mPFC GABA neurons Chefer et al., 2011). In the present study in-vivo microdialysis was used to identify alterations in rat mPFC glutamate, GABA dopamine and norepinephrine overflows in response to cues previously associated with either saline or cocaine in situations of either low or high certainty of cocaine expectations. Neurotransmitter levels and locomotor activity (LMA) were assessed 24 hrs after the last conditioning session. Some animals were stressed by handling immediately prior to testing. Exposure to a cocaine-associated environment following fixed sequences of conditioning trials increased mPFC GABA overflow and decreased Glutamate/GABA ratio. Handling stress, which increased blood corticosterone levels, increased baseline Glutamate and GABA overflow in rats expecting cocaine administration. Introduction of the cocaine cues augmented GABA overflow and decreased the Glutamate/GABA ratio in these rats. When random sequences of conditioning trials were used and the certainty of cocaine expectation therefore was low, the same stress paradigm did not increase basal GABA levels. Contrary to the fixed sequence regimen, glutamate overflow and Glu/GABA ratio was increased by cocaine cues. Saline-associated cues did not alter Glu or GABA during either trial regimen. Conditioned hyperactivity in cocaine environment was observed in high expectations paradigm and following stress in low expectations paradigm. To our knowledge, the present study is the first to demonstrate a neurochemical interaction between cocaine-associated environment, stress and cocaine expectation. The data suggest that changes in mPFC Glu/GABA equilibrium reflect certainty of cocaine administration and stress reveals the level of expectation before actual cue presentation (manuscript in preparation).